WO2022167891A1 - Procédé pour la régénération d'un matériau de chromatographie en phase inverse à base de silice - Google Patents

Procédé pour la régénération d'un matériau de chromatographie en phase inverse à base de silice Download PDF

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Publication number
WO2022167891A1
WO2022167891A1 PCT/IB2022/050579 IB2022050579W WO2022167891A1 WO 2022167891 A1 WO2022167891 A1 WO 2022167891A1 IB 2022050579 W IB2022050579 W IB 2022050579W WO 2022167891 A1 WO2022167891 A1 WO 2022167891A1
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Prior art keywords
regeneration
resin
chromatography material
solution
process according
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PCT/IB2022/050579
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English (en)
Inventor
Sathish ALAGURAJAN
Sandeep V KAMATH
Shabandri QAIS
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Biocon Biologics Limited
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Publication date
Application filed by Biocon Biologics Limited filed Critical Biocon Biologics Limited
Priority to KR1020237029939A priority Critical patent/KR20230142566A/ko
Priority to CN202280012939.2A priority patent/CN116806237A/zh
Priority to US18/275,356 priority patent/US20240116029A1/en
Priority to AU2022216492A priority patent/AU2022216492A1/en
Priority to CA3205577A priority patent/CA3205577A1/fr
Priority to EP22749311.1A priority patent/EP4263692A1/fr
Priority to JP2023546423A priority patent/JP2024505100A/ja
Publication of WO2022167891A1 publication Critical patent/WO2022167891A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/34—Regenerating or reactivating
    • B01J20/3433—Regenerating or reactivating of sorbents or filter aids other than those covered by B01J20/3408 - B01J20/3425
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/20—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the conditioning of the sorbent material
    • B01D15/203—Equilibration or regeneration
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/282—Porous sorbents
    • B01J20/283—Porous sorbents based on silica
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
    • B01J20/287—Non-polar phases; Reversed phases
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/34—Regenerating or reactivating
    • B01J20/345—Regenerating or reactivating using a particular desorbing compound or mixture
    • B01J20/3475—Regenerating or reactivating using a particular desorbing compound or mixture in the liquid phase

Definitions

  • the present invention relates to a process for the regeneration of a reverse phase chromatography material.
  • Purification of a polypeptide from a mixture is a step which is used several times during the overall manufacturing process for a therapeutic polypeptide.
  • the majority of biomolecules such as proteins and polypeptides are used for therapeutic purposes and need to undergo multiple purification steps to ensure utmost purity.
  • RPC reverse phase chromatography Due to the continuous use of the chromatographic resin, there is a build-up of left over proteins, mixed compounds and other impurities on the chromatographic resin. This build-up over time increases the pressure in the column and can affect retention time of the product and the column performance. Therefore the resin needs to be regenerated after a defined numbers of cycles to restore the resin for an optimal resolution of impurities. Due to the physio-chemical properties of the solutions, buffers and continual reuse of the resin, the life span of an RPC resin despite the regeneration process is minimal.
  • the RPC loads comprise Pichia pigments and inorganic salts that are a carryover from preceding processing steps. All these foreign components other than the peptide of interest slowly accumulate on to the resin, leading to high pressure, noncomphance to set in-process quality limits and eventually to pre-expiry of the resin.
  • Another method used for regeneration involves unpacking and repacking of a column between defined numbers of cycles in order to remove the pigmented resin and to replace it with fresh resin. As a consequence, resin is removed before its maximum usage. Further, frequent unpacking of the resin damaged silica particles and lead to defining of the resin that eventually chokes the column frits. Also, unpacking and repacking increased the operational time, and is not a feasible and recommended at commercial scale manufacturing.
  • U.S. Pat. No. 9,364,772 discloses a process to regenerate a stationary chromatography phase made of silica material or substituted silica material, such as C8, by using a regeneration solution comprising formic acid as a solvent.
  • WO 2007/050221 discloses a method for using chlorine dioxide, e.g., pure chlorine dioxide solution, for cleaning, regenerating and/or sterilizing chromatographic media.
  • Chinese patent publication no. 105289555 relates to a method for regeneration of a resin by removing the silica resin from assembly, addition of 2% sodium hypochlorite (soaking for 2hrs) followed by sodium hydroxide solution.
  • Chinese patent publication no. 101612598 discloses a method to resuscitate the activity of a contaminated resin by sodium hypochlorite after the third step along with an iron ion complexing agent and a dispersing agent for 2-3 hrs.
  • a process of regenerating a reverse-phase chromatography material Provided is a process of regenerating a reverse-phase chromatography material. Provided is also the use of a regeneration solution for regenerating a reverse-phase chromatography material.
  • the present process and the use are generally for cleaning or regenerating a reverse phase chromatography material, e.g., a reverse phase chromatography resin, after carrying out chromatography, for subsequent use.
  • a reverse phase chromatography material e.g., a reverse phase chromatography resin
  • the reverse phase chromatography material may be included in a chromatography column.
  • the reverse phase chromatography material may be included in a column in packed form.
  • the disclosure relates to the regeneration of used reverse phase chromatography material.
  • the process includes allowing a regeneration solution to pass through and/or over the chromatography material.
  • the regeneration solution is allowed to pass through and/or over the chromatography material upward from bottom of the resin.
  • applying the regeneration solution is followed by allowing water, e.g. purified water, to pass through the chromatography material.
  • a process for regenerating chromatographic resin using a regeneration solution comprising steps:
  • the sodium hypochlorite in solution is provided at an alkaline pH.
  • Denaturation of protein generally, occurs at alkaline pH, due to which conformation of protein changes and gets removed from the resin.
  • the ligands attached to the stationary phase e.g. the resin, are often vulnerable to alkaline pH, thus it is important to neutralize using acid at pH ⁇ 5.
  • traces of regeneration and neutralization buffer components used in the previous steps are removed with water and/or a solvent such as acetonitrile, until the outlet conductivity matches with the inlet conductivity.
  • FIG. 1 refers to procedure for Sodium hypochlorite regeneration
  • FIG. 2 depicts the resin tested with different reagents and solvents where it can be observed that use of only Sodium hypochlorite (4 th bottle from left) resulted in the removal of colour from the resin.
  • FIG. 3 depicts the standard curve obtained of the absorbance vs concentration of sodium hypochlorite during Rhodamine B analysis.
  • FIG. 4A refers to column test performed after Sodium hydroxide regeneration
  • FIG. 4B refers to column test performed after Sodium hypochlorite regeneration
  • FIG. 5A and 5B refers to RP-1 Cycle before Sodium hypochlorite regeneration and RP-1 Cycle after Sodium hypochlorite regeneration, respectively.
  • FIG. 6 illustrates the physical appearance of the resins at various stages (fouled, regenerated and fresh) and colour of the resins observed.
  • FIG. 7A-7E depicts the particle size of the silica gel resins analyzed by Sedimentation method.
  • FIG. 8 is chromatogram of Pyridine-Phenol test conducted for detecting Silanol activity, showing similarity in peak for fresh resin and regenerated resin.
  • chromatographic material e.g. a resin that has been used in preparative phases of purification of a therapeutic polypeptide such as recombinant human insulin, insulin analogues and derivatives.
  • the process for regenerating a chromatographic resin of reverse phase-high pressure liquid chromatography involves a step wherein said chromatographic resin is contacted with a regeneration solution, followed by neutralization and washing.
  • the regeneration solution passed through the resin from a bottom of the resin upward through the column without unpacking resin from the column.
  • the process is a process for regenerating a chromatographic resin for at least 30 minutes by contacting with regeneration solution, wherein, the said regeneration solution comprise of bleaching agent such as sodium hypochlorite and organic solvent such as Acetonitrile (ACN).
  • bleaching agent such as sodium hypochlorite
  • organic solvent such as Acetonitrile (ACN).
  • ACN Acetonitrile
  • Regeneration is in some embodiments followed by neutralization solution at acidic pH ⁇ 5.
  • the neutralization solution contains an organic acid such as acetic acid.
  • the neutralization solution also contains an organic solvent such as acetonitrile.
  • the traces of regeneration and neutralization buffer components used in the previous steps are cleaned. This may for example be done with water and acetonitrile. In some embodiments washing is carried out until the outlet conductivity matches with the inlet conductivity.
  • Another aspect of the present disclosure is the use of a process disclosed herein for regenerating a chromatographic resin. Thereby the pressure drop occurring over the length of the chromatographic column, when in use, is reduced.
  • a regeneration solution that contains a bleaching agent and an organic solvent for regenerating a reverse-phase chromatography material.
  • the bleaching agent is an oxidizing agent such as a hypochlorite, a chlorite, a chlorate or a perchlorate.
  • a hypochlorite include but are not limited to sodium hypochlorite (NaOCl), calcium hypochlorite potassium hypochlorite, hydrogen peroxide, chlorine dioxide and mixtures thereof.
  • NaOCl sodium hypochlorite
  • CaOCl calcium hypochlorite potassium hypochlorite
  • hydrogen peroxide chlorine dioxide and mixtures thereof.
  • chlorine dioxide sodium hypochlorite
  • NaOCl sodium hypochlorite
  • CaOCl calcium hypochlorite and mixtures thereof.
  • the word “about” as used herein refers to a value being within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within one, or more than one standard deviation, per the practice in the art. The term “about” is also used to indicate that the amount or value in question may be the value designated or some other value that is approximately the same. The phrase is intended to convey that similar values promote equivalent results or effects as disclosed herein. In this context “about” may refer to a range above and/or below of up to 10%.
  • the word “about” refers in some embodiments to a range above and below a certain value that is up to 5%, such as up to up to 2%, up to 1%, or up to 0.5 % above or below that value. In one embodiment “about” refers to a range up to 0.1 % above and below a given value.
  • chromatography material and “chromatographic material” as used herein mean the stationary solid phase over which the soluble phase passes, i.e. the chromatographic matrix.
  • the chromatographic material is typically arranged within a chromatographic column.
  • the chromatographic material may be packed into a column.
  • the material may be a resin.
  • Porous or nonporous silica particles with coupled hydrophobic moieties such as alkyl chains are often used in the art in reversed-phase chromatography.
  • the resin may be made of substituted silica, such as C-4 silica, C-8 silica, C-12 silica and C-18 silica.
  • the structure of silica is built up of a three-dimensional combination of the siloxane link Si— O— Si and the silanol link Si— OH.
  • a chromatographic material are a membrane, a monolithic material and a filter.
  • the chromatographic material may be defined by a monolithic column.
  • a monolithic column may include a plurality of channels arranged inside the column.
  • Reversed- phase polymeric monoliths are commercially available from BIA Separations (Agilent technologies) or Merck KGaA. Often a resin such as a resin consisting of beads is used.
  • a chromatography material used in reverse-phase chromatography is a hydrophobic material.
  • regeneration solution refers a solution which is used to regenerate a chromatographic resin.
  • the purpose of the regeneration is to keep a satisfactory performance of the chromatographic separation over several chromatographic cycles.
  • a regeneration step may comprise contacting of the chromatographic resin with either a single regeneration solution or with more than one regeneration solution.
  • organic solvent as used herein means a solvent which comprises at least one carbon-atom and which can be maintained in the fluid state for carrying out the process disclosed herein.
  • the organic solvent used is a solvent that is in the liquid state under atmospheric pressure conditions throughout the temperature range from 0° C to 50° C.
  • Non-limiting examples of organic solvents are lower alcohols such as methanol and ethanol, poly hydric alcohols, acetonitrile, hexane and acetone, ethanol, 1 -propanol, 2-propanol, and hexyleneglycol.
  • bleaching agent refers to a material that lightens or whitens a substrate through chemical reaction.
  • the bleaching reactions usually involve oxidative or reductive processes that degrade colour systems.
  • Non-limiting examples of bleaching agent include sodium hypochlorite, hydrogen peroxide (H2O2).
  • pharmaceutical composition means a product comprising an active compound or a salt thereof together with pharmaceutical excipients such as buffer, preservative and tonicity modifier, said pharmaceutical composition being useful for treating a disease or disorder.
  • pharmaceutical composition is also known in the art as a pharmaceutical formulation.
  • therapeutic polypeptide as used herein means a polypeptide for which there is a recognized potential utility as a therapeutic agent.
  • therapeutic polypeptides are human insulin, analogues and derivatives, thrombopoetin, erythropoietin and human growth hormone etc.
  • RPC refers to reverse-phase chromatography that uses a hydrophobic chromatographic resin.
  • the term mainly refers to liquid (rather than gas) chromatography.
  • Reversed-phase liquid chromatography separates molecules based on surface hydrophobicity. Since the resin is hydrophobic, molecules with hydrophobic properties contained within the mobile phase will have a high affinity for the resin and get adsorbed to the column packing. Elution of the hydrophobic molecules adsorbed to the column packing requires the use of more hydrophobic or more non-polar solvents in the mobile phase to shift the distribution of the particles in the resin towards that of the mobile phase.
  • RP-HPLC refers to reverse phase high pressure liquid chromatography which is a technique in analytical chemistry used for manufacturing, research and medical purposes to separate, identify and quantify each component in a mixture. It relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a column filled with a solid adsorbent material. Each component in the sample interacts slightly differently with the adsorbent material, causing different retention time for the different components and leading to the separation of the components as they flow out of the column.
  • Cold Volume refers to amount of mobile phase that can be contained in the chromatography column.
  • Pichia pastoris refers to a species of methylotrophic yeast which is frequently used as an expression system for the production of therapeutic polypeptides.
  • human insulin as used herein means the human hormone whose structure and properties are well known, and includes naturally occurring isoforms and variants.
  • the protein is generated by processing of preproinsulin, which can be found under Uniprot database entry PO13O8 (version 256 of 12 August 2020, version 1 of the sequence of 21 July 1986), of which isoform 1, the canonical sequence, has 110 amino acids.
  • variants which are often associated with a disease, include the variants having a mutation a mutation H — D at position 29, a mutation G — R at position 32, a mutation G — S at position 32, a mutation H — D at position 34, a mutation C — G at position 43, a mutation G — V at position 47, or a mutation S — C at position 101, to name a few.
  • Further variants can be taken from the above Uniprot entry.
  • Human insulin has two polypeptide chains that are connected by disulphide bridges between cysteine residues, namely the A- chain and the B -chain.
  • the A-chain is a 21 amino acid peptide, corresponding to amino acid positions 90 - 110 of preproinsulin
  • the B-chain is a 30 amino acid peptide, corresponding to amino acid positions 25 - 54 of preproinsulin.
  • the two chains are connected by three disulphide bridges: one between the cysteine in position 6 and 11 of the A-chain (positions 95 and 100 of preproinsulin), the second between the cysteine in position 7 of the A-chain and the cysteine in position 7 of the B-chain (positions 31 and 96 of preproinsulin), and the third between the cysteine in position 20 of the A-chain and the cysteine in position 19 of the B-chain (positions 43 and 109 of preproinsulin).
  • analogue as used herein in relation to a parent polypeptide means a modified polypeptide wherein one or more amino acid residues of the parent polypeptide have been substituted/deleted/added by other amino acid residues. Such addition or deletion or substitution of amino acid residues can take place at the N-terminal of the polypeptide or at the C-terminal of the polypeptide or within the polypeptide.
  • An insulin analogue is able to induce the same effects and have the same function in the human body in terms of controlling blood glucose levels as the naturally occurring canonical form of insulin.
  • Two examples of an insulin analogue are insulin Aspart and insulin Lispro.
  • Two further examples of an insulin analogue are insulin Glargine and insulin Glulisine.
  • an insulin analogue is Detemir insulin and Degludec insulin.
  • Yet another example of an insulin analogue is insulin tregopil.
  • Other examples are porcine or bovine insulin which can both be taken to be analogues of human insulin.
  • Aspart refers to rapid-acting insulin analogue which differs from human insulin by a single substitution of proline with aspartic acid in position 28 on the C-terminal end of the B-chain.
  • Glargine refers to long-acting insulin analogue which differs from human insulin wherein the amino acid asparagine at position 21 on the Insulin A- chain is replaced by glycine and two arginine residues are added to the C-terminus of the B chain.
  • Lepro refers to rapid-acting insulin analogue which differs from human insulin wherein lysine and proline residues at position 28 and 29 on the C- terminal end of the B -chain are reversed.
  • unbound, entrained and/or non- specifically adsorbed material refers to any unwanted material from a production batch of a wanted target (bio)molecule, such as mammalian cells, bacteria, fragments of cellular organisms, proteins, nucleic acids, lipids, etc. derived from cellular organisms, which can either degrade the immobilised adsorption ligands, block the ligands from interacting with the solution or physically block pores in the adsorbent so that target molecules cannot reach the immobilised adsorption ligands or pose a risk of crosscontamination between successive batches of produced target biomolecule reusing the same batch of adsorbent.
  • a wanted target (bio)molecule such as mammalian cells, bacteria, fragments of cellular organisms, proteins, nucleic acids, lipids, etc. derived from cellular organisms, which can either degrade the immobilised adsorption ligands, block the ligands from interacting with the solution or physically block pores in the adsorb
  • a typical chromatography process consists of 5 steps, as follows:
  • Loading or sample application Loading or sample application is the step where the protein mixture is loaded onto the column for binding.
  • Washing performed to remove the unbound or weakly bound proteins from the column.
  • Regeneration performed to remove the strongly bounded protein.
  • An additional regeneration of the column is performed to remove the strongly bounded molecules, pigments and buffer components, which cannot be removed by chromatography regeneration step.
  • the process disclosed herein relates to this additional regeneration of the column which is carried out after the chromatography regeneration step.
  • the polypeptide purification process is typically an elution chromatography process.
  • the regeneration process can in this case also be taken to be an elution chromatography process, with the difference that in regeneration left-over matter that is undesired is to be eluted from the chromatography material.
  • the compound of interest may be a polypeptide such as a therapeutic polypeptide product.
  • a therapeutic polypeptide product is generally manufactured by a process comprising the steps of:
  • a process as disclosed herein includes contacting a chromatography material that is to be cleaned with a regeneration solution.
  • the chromatography material is the stationary phase used in the chromatography process.
  • the chromatography material may define a bed along or through which the mobile phase can be allowed to flow.
  • the chromatography material may be a resin.
  • the chromatographic material may be any reversed phase material such as an RP-HPLC matrix.
  • a chromatographic resin used is a HPLC resin.
  • a resin for RP-HPLC may be silica such as C4- or C6-silica.
  • the chromatographic material may be C8- or ClO-silica.
  • the chromatographic material may in some embodiments be C12- or C16-silica.
  • the chromatographic material may also be C18- or C30- silica.
  • the chromatographic material may be cyclohexylsilica or phenyl silica.
  • the chromatographic material may in some embodiments be phenyl propyl silica or phenyl-butyl silica. In some embodiments the chromatographic material may be phenyl-hexyl silica or a pentafluorophenyl silica. In some embodiments the chromatographic material may be a cholesteryl silica or a cyano silica. Suitable silica material for use as chromatographic resin are for example spherical particles with a narrow pore size and particle sizes in the range from 3 pm to 100 pm. In some embodiments the pore size is in the range of 60 A to 300 A, such as 100 A or 120 A. The pore size may also be 150 A or 175 A. In some embodiments the pore size may be 200 A or 300 A.
  • the chromatographic column can be packed with the resin.
  • the column diameter is in the range of 3 to 1200mm. In some embodiments the column has a diameter of 4.6mm or 10mm. In some embodiments the column has a diameter of 21.2mm or 50mm. In some embodiments the column has a diameter of 400mm or 600mm. In some embodiments the column has a diameter of 800mm.
  • the regeneration solution with which the chromatography material is contacted, contains a bleaching agent and an organic solvent.
  • the process typically includes allowing the chromatography material to contact the regeneration solution for a certain period of time. During this period of time the regeneration solution may be in a state of flow or in a static state.
  • Suitable bleaching agents that may be included in the process disclosed herein have already been disclosed above.
  • a solvent present in the mobile phase in reversed-phase chromatography the mobile phase is substantially more polar than the stationary phase. Therefore, for the purification of a polypeptide, a polar solvent is included in the mobile phase.
  • a less polar mobile phase may be used in order to be able to elute matter that has bound to the stationary phase and that cannot, or not completely, be removed under the conditions used for purification purposes.
  • An organic solvent of low or medium polarity, or even a non-polar solvent may thus be used, as long as its use does not lead to a detrimental effect on the stationary phase.
  • the organic solvent included in the regeneration solution may in some embodiments be 2-methoxyethanol or A,A-di methyl formamide.
  • the organic solvent may be dimethyl acetamide or N- methylpyrrolidone.
  • the organic solvent may be dimethyl sulfoxide or acetonitrile.
  • the organic solvent may be 3- pentanol or 2-pentanol.
  • the organic solvent may be i- butanol or aniline.
  • the organic solvent may have a dielectric constant in the range from 40 to 5.
  • acetic acid has a dielectric constant of 6, and isopropyl alcohol has a dielectric constant of 18.
  • dichloromethane has a dielectric constant of 9.1.
  • tetrahydrofuran has a dielectric constant of 7.5 and hexamethylphosphoric triamide has a dielectric constant of 30.
  • benzonitrile with a dielectric constant of 25.9 and methyl ethyl ketone with a dielectric constant of 18.5.
  • the organic solvent may also have a dielectric constant in the range from 5 to 1.5.
  • hexane has a dielectric constant of 1.9
  • 1,4-dioxane has a dielectric constant of 2.3.
  • the chromatography material is contacted with the regeneration solution while the chromatography material is arranged within a chromatography column. In some embodiments the chromatography material is contacted with the regeneration solution without unpacking the resin from a column. Where desired, the process may also be carried out in a different column than used for a preceding purification process or outside a chromatography column. In some embodiments the chromatography material is contacted with the regeneration solution after unpacking the resin from a column.
  • the regeneration solution, with which the chromatography material is contacted is in some embodiments alkaline.
  • the regeneration solution has a pH of 10 or more or a pH of 11 or more.
  • the regeneration solution has a pH of 12 or more.
  • the chromatography material may be allowed to contact the regeneration solution for any desired period of time.
  • a contact time with the regeneration solution may be selected to be in the range from 1 minute to 8 hours or more, such as up to 300 minutes. In some embodiments a contact time from 5 minutes to 120 minutes, or from 10 minutes to 60 minutes may be chosen.
  • the chromatography material may be allowed to contact the regeneration solution for 30 minutes or for 45 minutes.
  • Contacting the chromatography material with a regeneration solution may be carried out by allowing a volume of a half or of one or more material volumes of the column to pass over and/or through the chromatography material. In some embodiments two or more or three or more material volumes are allowed to pass over and/or through the chromatography material.
  • the process disclosed herein also includes contacting the chromatography material with a neutralization solution. Thereby the chromatography material is neutralized. Contacting the chromatography material with a neutralization solution is done after allowing the chromatography material to contact the regeneration solution. Therefore, the chromatography material is at this stage in a regenerated state.
  • the neutralization solution contains an organic acid and an organic solvent.
  • any organic acid may be used in the neutralization solution, as long as no detrimental reaction with the bleaching agent is allowed to occur and as long as care is taken no undesired acidification is allowed to proceed. Depending on the subsequent process or storage that may be intended, an acidification may not be desired.
  • the organic acid is a carboxylic acid.
  • the organic acid is a carboxylic acid of two or more carbon atoms.
  • the organic acid has a pKa value in the range from 1.5 to 5.0, such as in the range from 2.5 to 5.0.
  • the organic acid is acetic acid, having a pKa of 4.76, or butanoic acid, having a pKa of 4.82.
  • the organic acid is 2-hydroxy-benzoic acid, having a pKa of 2.97, or hydroxy ethanoic acid, having a pKa of 3.88. In some embodiments the organic acid is 4-hydroxy-benzoic acid, having a pKa of 4.58, or lactic acid, having a pKa of 3.86. In some embodiments the organic acid is oxalic acid, having a second pKa value of 4.14.
  • the organic acid is different from formic acid. In some embodiments the organic acid is formic acid.
  • the regenerated chromatographic resin is contacted with a neutralization solution that includes an organic acid and an organic solvent.
  • the organic solvent included in the neutralization solution may be an organic solvent described above for the regeneration solution.
  • the organic solvent included in the regeneration solution and the organic solvent included in the neutralization solution may be selected independent from each other.
  • the column need not be unpacked to perform this step.
  • the neutralization can be performed inside the chromatography column.
  • the organic acid is acetic acid, such as 0.5 to 17.5 M acetic acid.
  • the organic solvent is acetonitrile, such as 100% pure acetonitrile. Ligands attached to the stationary phase are often vulnerable to alkaline pH, in which case it is desirable to neutralize using acid at pH ⁇ 5.
  • any purification process may precede the regeneration process.
  • the purification process may have involved or concerned a therapeutic polypeptide.
  • the therapeutic polypeptide may for instance be selected from the group consisting of human insulin, a human insulin precursor, a human insulin analogue, a human insulin analogue precursor, and derivatives thereof.
  • the polypeptide may be selected from the group consisting of recombinant human insulin, insulin Glargine, insulin Aspart, insulin Lispro, and insulin Tregopil etc.
  • the chromatographic resin in a first step, is contacted with a regeneration solution that contains one or more bleaching agent(s) and one or more organic solvent(s).
  • Bleaching agent such as sodium hypochlorite solution was used at alkaline pH. Denaturation of protein occurs at alkaline pH and gets removed from the resin.
  • the bleaching agent or oxidizing agent is sodium hypochlorite, for example in the range of 04-4.0%.
  • the organic solvent is acetonitrile.
  • a typical example of a bleaching agent that may be included in the regeneration solution is sodium hypochlorite. It is illustrated to remove colour from the resin employed. The mechanism of colour removal in the example below is due to the oxidation of either Mg 2+ or Fe 2+ from the phytochromobilin, which is a pigment produced by the Picha pastoris and higher pH of the sodium hypochlorite removed all the bounded particle from the resin.
  • the chromatographic resin may be contacted with the regeneration solution inside the chromatographic column. In this way a minimum of production capacity is lost due to down-time in connection with the regeneration step. Thus, the process of regenerating the chromatographic resin can be performed without repacking the column.
  • the chromatographic material may be contacted with the regeneration solution until the pH of the chromatography resin reaches a value of 11 or more.
  • Denaturation of protein generally, occurs at alkaline pH and gets removed from the resin.
  • a flow of regeneration solution over and/or through the chromatography material is allowed, until the pH of liquid that leaves the chromatography material reaches a value of 11 or more.
  • a flow of regeneration solution over and/or through the chromatography material is allowed to occur for 2 or more column material volumes.
  • contacting the chromatography resin with a regeneration solution sodium hypochlorite and acetonitrile until the fluid leaving the column reaches a pH of >11 is run for at least 2 or more additional column volumes.
  • contacting the chromatography material with a regeneration solution is carried out by allowing one or more column volumes to pass over and/or through the chromatography material.
  • contacting the chromatography material with a regeneration solution is carried out by allowing 3 or more column volumes, such as 4 or more column volumes to pass over and/or through the chromatography material.
  • the column need not be unpacked to perform this step.
  • the process can be performed inside the chromatography column.
  • Contacting the chromatographic material with the regeneration solution may in some embodiments be performed in a temperature range from 5 to 40 °C. In some embodiments contacting the chromatography material with the regeneration solution may be carried out in the range from about 20° C to 30° C. In one embodiment the chromatography material is contacted with the regeneration solution at room temperature, e.g. 25 °C.
  • the process disclosed herein also includes washing the chromatography material with a water containing solution. At this stage the chromatography material is regenerated and neutralized.
  • the chromatographic material is typically contacted with a wash solution that contains purified water and an organic solvent. Any organic solvent may be used in the wash solution, as long as the organic solvent is compatible with the chromatography material. It may in some cases be desirable to choose an organic solvent that corresponds to an organic solvent that is intended to be used after the process of regenerating the chromatography material. It may in some cases also be desirable to choose an organic solvent that has been found to be suitable for storage under desired conditions.
  • the organic solvent included in the wash solution is an organic solvent as described above for the regeneration solution. In some embodiments the organic solvent included in the wash solution is an organic solvent as described above for the neutralization solution.
  • the organic solvents included in the regeneration solution, in the neutralization solution and in the wash solution are selected independent from each other. In some embodiments the organic solvent included in the wash solution is acetonitrile, such as 100% pure acetonitrile.
  • the water included in the wash solution may in some embodiments be purified water.
  • washing may be allowed to occur until the conductivity of the wash solution before and after contacting the chromatography material remains unchanged.
  • a column containing the chromatography material may for instance be washed until the outlet conductivity matches with the inlet conductivity. The column need not be unpacked to perform this step.
  • the washing can be performed inside the chromatography column. In some embodiments washing the neutralized chromatography material is carried out inside a chromatography column. In some embodiments washing the neutralized chromatography material may be carried out outside a chromatography column.
  • contacting the chromatography material with a wash solution is carried out by allowing one or more column volumes to pass over and/or through the chromatography material. In some embodiments contacting the chromatography material with a regeneration solution is carried out by allowing two or more column volumes, such as three or more column volumes to pass over and/or through the chromatography material.
  • the process of regeneration of a chromatographic resin, the third step of washing the chromatography resin with purified water and acetonitrile is carried out for 3 column volumes or for 5 column volumes.
  • the process for regenerating a chromatographic material as disclosed herein is applied to the chromatographic material after every 5 to 40 cycles of use in purification.
  • the process for regenerating is carried out for at least once every 50 chromatographic purification cycles, or at least once every 100 chromatographic purification cycles.
  • critical performance related parameters are typically the pressure and the number of theoretical plates over the chromatographic column.
  • Pressure and theoretical plates during column integrity test were evaluated by the present inventors to observe the effect of regeneration according to the process disclosed herein.
  • High pressure during column testing and a lower number of theoretical plates indicate that the resin is fouled, and that its condition may impact the resolution in the preparative (purification) cycles.
  • the pressure on a column tends to increase due to strong binding of protein/pigments in resin pores, clogging of pores due to strong binding of buffer components to resin etc.
  • pressure of more than 2.0 MPa and a number of plates less than 15000 N during column integrity testing is considered as high pressure on a column.
  • Table 2 Elaborates the comparative experimental data for all 6 chemicals tested for regeneration.
  • UOM unit of measurement
  • Sodium hydroxide, Acetic Acid (CH3COOH), Guanidine hydrochloride (GuHCl), Hydrochloric acid (HC1), Formic Acid (CH2O2) and Sodium hypochlorite (NaOCl) were tested for regeneration of chromatography resin.
  • the chromatography resin was a prepacked kromasil C8 silica column, which was failing for pressure during column test (More than 2.0MPa). High pressure during column test and lower number of plates indicates that the resin was fouled and it might have impacted the resolution on the preparative cycles.
  • the chemicals at alkaline pH greater than 11 denatures strongly bound proteins, nucleic acid, lipids, bacteria, and metal ions but is not observed to remove the pigment from resin.
  • IM Hydrochloric acid HC1
  • GuHCl Guanidine hydrochloride
  • IPA Iso-propyl alcohol
  • NaOCl 0.8% Sodium hypochlorite
  • C7H8 100% Toluene
  • 70% ACN Alcohol
  • Table 3 elaborate the colour of supernatant. It was observed that only sodium hypochlorite (4 th bottle from left in FIG. 2) resulted in the removal of colour from the resin. Sodium hypochlorite on dissociation releases the hypochlorite (OC1 ) ion, which is a strong oxidizing agent that oxidise the metal ion present in the pigment, thus removing all the pigmentation present in the column. Sodium hypochlorite in combination with higher pH ensures that all other contaminants are removed from the resin.
  • OC1 hypochlorite
  • Residual sodium hypochlorite present in the wash sample was quantified with spectrophotometric method using Rhodamine B. This method has been applied for the determination of sodium hypochlorite in its pure form.
  • a column test was conducted to measure the reduction in pressure after regeneration with sodium hydroxide (FIG. 4A) and sodium hypochlorite (FIG. 4B). Table 6 shows results of the column test.
  • FIG. 5A A column integrity test conducted to measure reduction in pressure before regeneration with sodium hypochlorite (FIG. 5A) and after regeneration with sodium hypochlorite (FIG. 5B). The regeneration was conducted as described in FIG. 1. This regeneration was conducted to evaluate the performance of preparative cycle before and after regeneration with sodium hypochlorite.
  • the regeneration results are depicted in FIG. 5 A, FIG. 5B and table 7.
  • the elution pressure during RP-1 cycle reduced after Sodium hypochlorite regeneration and start B% (the point at which elution collection starts which indicate retention of protein) were compared between before and after Sodium hypochlorite regeneration. It was observed that elution pressure reduced almost 50% and the elution start B% (retention of protein) also increased after sodium hypochlorite regeneration.
  • FIG. 5A shows RP-1 Cycle before Sodium hypochlorite regeneration while FIG. 5B shows RP-1 Cycle after Sodium hypochlorite regeneration.
  • the column was regenerated with mixture of 40% of 25mM sodium hydroxide and 60% of acetonitrile in up flow direction till the pH reaches >11 and continue to run for one more CV thereafter. During the first CV, the linear flow rate was maintained at ⁇ 100 Cm/h and Second CV onwards, the linear flow rate was maintained at ⁇ 220 cm/h. 2. The column was neutralized with mixture of 30% of 1 M acetic acid and 70% of acetonitrile in up flow direction for three CV at flow rate ⁇ 220 cm/h.
  • the column was regenerated with mixture of 40% of (0.8-2.0) % sodium hypochlorite and 60% of acetonitrile in up flow direction till the pH reaches >1 1 and continued to run for two more CV thereafter.
  • the linear flow rate was maintained at ⁇ 100 Cm/h and Second CV onwards the linear flow rate was maintained at ⁇ 220 cm/h.
  • the column was neutralized with mixture of 30% of 1 M acetic acid and 70% of acetonitrile in up flow direction for three CV at flow rate ⁇ 220 cm/h.
  • hypochlorite has a yield of 76.0% which shows that the performance of cycles improved after hypochlorite regeneration.
  • Particle size was analyzed by Sedimentation method [Average D50 (pm), Distribution D40/D90] using Centrifugal Automatic Particle Analyzer CAPA-300 (Horiba, Ltd.) as Sedimentation-type particle size analyzer.
  • Particle size of silica gel as a raw material was measured by it as follows.
  • D50 50% accumulative weight
  • the particle size of the resins A, B, C, D and E were observed as depicted in FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D and FIG. 7E respectively.
  • Table 11 elaborate the average particle size of each resin.
  • Pore size, Surface area and Pore volume of silica gel as a raw material were measured by B.E.T. method with AUTOSORB-l-Kr (Sysmex Corp.) as follows.
  • the slight pore size increase after regeneration treatment may indicate the clearing of pores which may have been fouled by impurities.
  • Post regeneration treatment the pore sizes were comparable to that of the negative control resin E. No damage was attributed to regeneration treatment.
  • the pore volumes were observed to increase slightly after the regeneration treatment. This may indicate any pores which might have been blocked by the impurities has been cleared post the regeneration treatment.
  • Carbon, Hydrogen and Nitrogen content of the modified silica gel was measured by CHN coder (Yanaco Co.). Modified part of silica sample was decomposed to H2O, CO2 and N2 by perfect combustion and their concentrations were measured as follows:
  • the elemental analysis as shown in table 15 shows that the regeneration treatment was capable of bringing down the %C, %H and %N to basal levels.
  • the basal level of %C and %H is contnaded by the ligands of the resin.
  • the decrease in %C, %H and %N by 0.8%, 0.1% and 0.26% respectively which may be attributed to impurities of biological nature which was removed as a result of regeneration treatment.
  • the Pyridine-Phenol test is for detecting Silanol activity.
  • silica extended usage with repeated NaOH wash
  • small parts of the silica particles break off.
  • the silica surface goes through Silanol deactivation by the bonding of the (C8) ligands and the end-capping.
  • Silanol groups get exposed.
  • the peak for Pyridine starts getting lesser retention time indicative of loss of ligands which is undesirable and the peak for Phenol shows increase in retention time due to the increased Silanol Activity which is also undesirable.
  • Table 16 shows the results of Pyridine-Phenol test, from which, it was observed that upon regeneration treatment the values are fairly consistent indicating no loss of ligands or increase in silanol activity. Hence the column performance was not impacted by the regeneration treatment carried out. As seen in FIG. 8, the peak obtained for fresh resin and resin regenerated after use are similar, thus, we concluded the column performance was not impacted by the regeneration treatment carried out.
  • the process of regeneration of chromatography resin of present invention was 10 able to regenerate resin therefor proved useful to make the maximum use of resin, reduce the loss of resin and unpack repack frequency. It also increased the resin life time, which proves to be a commercial advantage at manufacturing. Similar process can be used for chromatography column used for other insulin analogues such as Lispro, Aspart and recombinant human insulin.

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  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • Treatment Of Liquids With Adsorbents In General (AREA)
  • Solid-Sorbent Or Filter-Aiding Compositions (AREA)
  • Peptides Or Proteins (AREA)
  • Silicon Compounds (AREA)

Abstract

La présente invention concerne un procédé pour la régénération de la résine de silice usée utilisée en chromatographie sans déballer la résine de la colonne de chromatographie. Le procédé comprend 3 étapes, à savoir : la mise en contact de la résine de chromatographie avec une solution de régénération, la neutralisation de la résine de chromatographie régénérée avec une solution de neutralisation et le lavage de la résine de chromatographie neutralisée avec une solution de lavage.
PCT/IB2022/050579 2021-02-02 2022-01-24 Procédé pour la régénération d'un matériau de chromatographie en phase inverse à base de silice WO2022167891A1 (fr)

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KR1020237029939A KR20230142566A (ko) 2021-02-02 2022-01-24 실리카 기반 역상 크로마토그래피 물질의 재생 공정
CN202280012939.2A CN116806237A (zh) 2021-02-02 2022-01-24 二氧化硅基反相色谱材料的再生方法
US18/275,356 US20240116029A1 (en) 2021-02-02 2022-01-24 A process for the regeneration of silica based reverse phase chromatography material
AU2022216492A AU2022216492A1 (en) 2021-02-02 2022-01-24 A process for the regeneration of silica based reverse phase chromatography material
CA3205577A CA3205577A1 (fr) 2021-02-02 2022-01-24 Procede pour la regeneration d'un materiau de chromatographie en phase inverse a base de silice
EP22749311.1A EP4263692A1 (fr) 2021-02-02 2022-01-24 Procédé pour la régénération d'un matériau de chromatographie en phase inverse à base de silice
JP2023546423A JP2024505100A (ja) 2021-02-02 2022-01-24 シリカベース逆相クロマトグラフィー材料の再生のためのプロセス

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6248683B1 (en) * 1999-04-07 2001-06-19 Silicycle Inc. Process for the regeneration of used silica gel
US9364772B2 (en) * 2003-04-08 2016-06-14 Novo Nordisk A/S Regeneration of chromatographic stationary phases

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6248683B1 (en) * 1999-04-07 2001-06-19 Silicycle Inc. Process for the regeneration of used silica gel
US9364772B2 (en) * 2003-04-08 2016-06-14 Novo Nordisk A/S Regeneration of chromatographic stationary phases

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MAJORS R. E.: "The Cleaning and Regeneration of Reversed Phase HPLC Columns", LCGC NORTH AMERICA, vol. 21, no. 1, 1 January 2003 (2003-01-01), pages 19 - 24, XP002293699 *

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CN116806237A (zh) 2023-09-26
US20240116029A1 (en) 2024-04-11
AU2022216492A1 (en) 2023-07-27

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